Diabetic cardiomyopathy: does it exist ? New insights with Apolipoprotein O: Upregulated in human diabetic heart and promotes mitochondrial dysfunction and lipotoxicity. Dr Fatima SMIH TEAM 7: « Obesity and heart failure: molecular and clinical investigations » I2MC INSERM UMR 1048 1 Avenue Jean Poulhes BP 84225 31432 Toulouse cedex 4. France http://www.i2mc.inserm.fr.
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Diabetic cardiomyopathy: does it exist ?New insights with Apolipoprotein O: Upregulated in human diabetic heart and promotes mitochondrial dysfunction and lipotoxicity.
Dr Fatima SMIH
TEAM 7: « Obesity and heart failure: molecular and clinical investigations » I2MC INSERM UMR 1048
1 Avenue Jean Poulhes BP 84225 31432 Toulouse cedex 4. France http://www.i2mc.inserm.fr.
Global Projections for the diabetes epidemic: 2010–2030 => Impact on HEART ?
Data mining on 107 human heart microarrays from GEO database
Cardiac myoblasts cell lines overexpressing APOO
+ cDNA APOO A1/A4/E domain
1981
N-
40 83
-CWT APOO
H9c2 cardiomyoblasts
Expression vectors APOO
Control
Cont. APOO0
5
10
15*
FAM
E x
100
(nm
ol/m
g pr
otei
n)
APOO overexpression promotes lipid overload in cardiac myoblasts
= APOO Cells
CAPOO
Mit.CAPOO
Recom.APOO
APOO
ANT
Cyto.
APOO
Green MitotrackerAPOO/TMR stain Merge
APOO
Recom.APOO
Membranes APOO C
CALR.
What is APOO localization in cardiac myoblasts?APOO Overexpression
APOO:Fluorescentlabelling
Subcellular protein fractionation
Recom.APOO D 1-40 Cyto. Mit.
APOO D 1-40
ANT
ACTIN
APOO D1-40
Green MitotrackerAPOO/TMR stain Merge
APOO localizes to the mitochondria in cardiac cells
APOO D1-40 Overexpression in cardiac myoblasts
APOO D1-40:Fluorescent labelling
A1/A4/E domain
198
N-
40 83
-C ApoO Δ1-40
1
Mutated APOO
Deletion of the mitochondrial adress label
Subcellular protein fractionation
Control
shRNA APOOAPOO D 1-40APOOO 2
Flo
w(p
mol
/s/1
06 cel
ls)
0
25
50
75
100
*
**
Cont.0
1
2
3
4
5
Cyt.
C ox
yd. a
ctiv
ity(µ
mol
/min
/µg
prot
ein) ***
APOO0
500
1,000
1,500
2,000
2,500 ControlApoO
DCFDA (µM)DC
F flu
o/10
6 c
ells
0 1 2 3 4 5 60.0
0.1
0.2
0.3
Cyt.
C ox
yd. a
ctiv
ity(µ
mol
/min
/µg
prot
.)
APOO-TgWT
*
A role for APOO in mitochondrial function?
APOO enhances mitochondrial respiration and oxidative stress in cardiac myoblast.
0
2
4
6
8**
***
Cd36
mRN
A (A
.U.)
0
4
8
12
16
****
Fatp
4 m
RNA
(A.U
.)
0
2
4
6
8
10 *******
Nad
h dh
mRN
A (A
.U.)
0
2
4
6
8 *****
**
Ndu
fa7
mRN
A (A
.U.)
A role for APOO in mitochondrial function?
OxidativePhosphorylationgenes
Fatty acids transportersgenes
Control
shRNA APOOAPOO D 1-40APOO
0
10
20
30
40 ***
Palmitate
**Di
glyc
erid
es(n
mol
/mg
prot
ein)
Mouse heartCardiac myoblasts
APOOControl
APOO D1-40
WT APOO-Tg0
2
4
6
Digl
ycer
ides
(nm
ol/m
g pr
otei
n) **
WT APOO-Tg0
5
10
15
20
Trig
lyce
rides
(nm
ol/m
g pr
otei
n)
Palmitate0
10
20
30
40
Triglycerides
(nmol/mg protein)
0 0.2 0.4 0.60
2
4
6
8 r = 0.6389 P = 0.0002
APOO mRNA (A.U.)
Digl
ycer
ides
(nm
ol/m
g pr
otei
n)
Human heart
0
100
200
300 r = 0.0331 P = 0.8978
APOO mRNA (A.U.)
Trig
lyce
rides
(nm
ol/m
g pr
otei
n)0 0.2 0.4 0.6 0.8
APOO induces lipotoxicity
What happens when lipid uptake exceeds mitochondrial oxidative capacity?
0
2
4
6
8
10
12
****
Bax
mRN
A (A
.U.)
APOOCont. shAPOO
Cardiac myoblasts
shAPOO0
10
20
30**
***
Casp
ase
3 ac
tivity
x10
(RFU
/µg
prot
ein)
APOOCont.2
0 125 250 500 0
100
200
300
400
500
**
******
Casp
ase-
3 ac
tivity
x 1
O2
(RFU
/µg
prot
ein)
*
Palmitate
ControlAPOO
Human heart
0.2 0.4 0.6 0.80
0.1
0.2
0.3 r = 0.7289p < 0.0001
BAX
mRN
A (A
.U.)
APOO mRNA (A.U.)
Mouse heart
0
5
10
15***
Casp
ase
3 ac
tivity
x 1
O R
FU/µ
g pr
otei
n
APOO-TgWT
2
WTBax/
Bcl-2
mRN
A (A
.U.)
APOO-Tg
Is APOO inducing lipoapoptosis?
APOO induces apoptosis
• PPAR is a transcription factor involved in lipid uptake.
• Ppara overexpression in mice led to cardiac dysfunction.
• The expression and activity of PPAR increase in diabetic hearts.
• The increase in intracellular lipid content in APOO models, should be associated with an induction of the expression of PPAR
Are APOO models mimicking diabetic cardiomyopathy?
Finck BN, et al. J Clin Invest. 2002
APOO associates with an increase in Ppar expression.
Cont.APOO0
2
4
6
8
10
12
Ppar
a m
RNA
(A.U
.) *
P-AMPK
CALR
AMPK
Cont. APOO
Mouse heart
5 10 15 20 250
2
4
6
8 r = 0.81 P < 0.0001
Apoo mRNA (A.U.)
Ppar
a m
RNA
(A.U
.)
0 0.2 0.4 0.6 0.80
0.1
0.2
0.3
0.4
0.5 r= 0.48
PPAR
A m
RNA
(A.U
.) P = 0.0006
APOO mRNA (A.U.)0
Human heart
Cardiac myoblasts
• PGC-1 , a master regulator of mitochondrial biogenesis, is upregulated in diabetic heart
• Ultrastructural analyses have revealed mitochondrial damage in mouse models of the metabolic syndrome and in models of type 1 and type 2 diabetes.
Are APOO models mimicking diabetic cardiomyopathy?
Handschin C. et al. Endocr. Rev. 2006
Mitochondrial degradation in APOO cells
0 5 10 15 20 250
2
4
6
8r = 0.81 P < 0.0001
Apoo mRNA (A.U.)
Pgc1
a m
RNA
(A.U
.)
Mouse heart
Human heart
0 0.2 0.4 0.6 0.82
4
6
8
10
12 r= 0.63 P = 0.0002
mRN
A (A
.U.)
PGC-
1A
APOO mRNA (A.U.)
nM
nM
nMN
Control +100µM palmitate
aM
mf
mFN
APOO + 100µM palmitate
mf
mF
0.0
0.5
1.0
1.5
2.0
2.5
***
ATP/
AMP
ratio
Cont. APOO
PPARPPAR
Multilamellar bodies
ROSFATPs
LCFAs
NADHFADH2
Metabolic sink
b-Oxid.
Plasma membrane
1
2
4
3
Lipotoxic byproducts
5
DNA
PGC1PPAR
Nucleus
6
OxidativePhospho.
Cytoc.C
Cell death
BAX
Apoptosome
7
IMM
OMM
APOO
Conclusion: The pathological overexpression of APOO induces a mitochondrial metabolic sink
APOO overexpression drives the cell into a vicious cycle consisting of a cascade of maladaptive events that ends in apoptosis and cell death
mimicking the diabetic cardiomyopathy.
Turkieh et al. The Journal of Clinical Investigation, 2014
INSERM UMR 1048 Team 7
Toulouse University Hospital
Dr Philippe Rouet, PhDProf. Galinier, MD-PhDProf. C. Dambrin, MD-PhDDr P. Massabuau, MDDr M.Berry, MDM. BarutautM. F. EvaristiDr C. Caubère, Dr A. Turkieh,